The growing demand for antibodies accompanied by rapid development of antibody drugs has increased the demand for higher antibody purification efficiency and thus higher antibody binding capacity for an affinity gel support used for antibody purification with the immobilized immunoglobulin-binding protein. For immobilization of an immunoglobulin-binding protein on an insoluble support, reactivity of side chains of its amino acid residues are utilized. In order to increase the immunoglobulin binding amount, there have been various attempts to orientationally immobilize protein.
The present inventors have invented an immunoglobulin-binding protein with an orientationally controllable immunoglobulin-binding domain. Specifically, the present inventors selected a C domain with high alkali stability from the five immunoglobulin-binding domains of Staphylococcus protein A gene, and modified parts of its amino acid sequence to allow the protein to be disposed in such an orientation that the immunoglobulin binding site will not be blocked, and the binding of the protein to an immunoglobulin will not be inhibited when the protein is immobilized on a support via one or more lysine residues (PTL 1).
There is also an attempt to achieve oriented single-point immobilization by introducing cysteine to the C-terminus of a protein, and immobilizing the protein on a gel support via a disulfide bond (NPL 1) or a thioether bond (PTL 2). In another attempt, immobilization is controlled with the N-terminal α-amino group (PTL 3) or the C-terminal carboxyl group (PTL 5) of an immunoglobulin-binding protein that has had its lysine residues substituted with some other amino acid.
The previous report introducing a cysteine residue to C-terminus, and the report using a C-terminal carboxyl group or an N-terminal amino group for oriented, single-point immobilization achieve increased immunoglobulin G (IgG) binding amounts. However, it remains elusive as to the optimum number of monomers (domains) linked to construct an immunoglobulin-binding domain multimer. For example, NPL 1 produces a monomer, a dimer, and a pentamer that are immobilized on thiopropyl sepharose at one location via an S—S bond with the cysteine introduced to the C-terminus of a Z domain modified from the B domain of protein A. However, the support immobilizing the pentamer is described as yielding the same IgG binding amount as the dimer-immobilized support. NPL 2 produces a multimer of four linked B domains of protein A. However, it is reported that the tetramer had essentially the same activity as that of native-form protein A (SPA) of five binding domains, and was no different from the pentamer. This paper measures the precipitation levels of the complex formed, but does not evaluate the multimer with regard to immobilization on a gel support and its binding capacity. NPL 3 produces a monomer, a dimer, a pentamer, and a decamer of a Z domain. However, while these are shown to have IgG binding activity, the proteins are not immobilized on a gel support, or not measured for IgG binding on a support.
In PTL 4, the C-terminal carboxyl group is orientationally immobilized. This publication describes a dimer with two linked domains as having about the same level of binding strength as heteropentameric native-form protein A. PTL 2 successfully produces a support of high immunoglobulin binding capacity by introducing cysteine to the C-terminus of the amino acid sequence of pentameric native-form protein A, and orientationally immobilizing the protein via a thiol group. However, this publication does not give any consideration to multimers of order higher than pentamers. PTL 2 describes the pentamer as having an IgG molar binding ratio of 2 to 3. PTL 3 and PTL 5 describe repeating 2 to 5 binding domains, but do not clearly state the optimum number of repeats. PTL 6 in paragraph [0033] describes a tetramer as being most desirable among the dimeric to pentameric multimers of an alkali-stabilized Z-domain variant.
It has been previously reported that the number of repeating immunoglobulin-binding domains is desirably 2 to 5. However, there is no report that indicates the usefulness of multimers of more than five domains compared to native-form protein A having five domains. Specifically, it has been unclear as to the optimum number of binding domains that maximizes the immunoglobulin-binding activity when a multimer with more than one binding domain is immobilized on a support at the terminal portion, irrespective of the chemical reaction used to immobilize the protein.